The present invention relates to aqueous-based formulations of ionophore growth factors. More particularly, disclosed are encapsulated ionophore growth factors, a method of their production, and methods of treatment using encapsulated ionophores. The ionophore growth factors are encapsulated within nonphospholipid lipid vesicles which are themselves dispersed or suspended in an aqueous-based solution.
Ionophore growth factors are primarily macrolide antibiotics that attack a variety of Gram-negative bacteria and increase growth rate, particularly of ruminant animals. The ionophores are active in ruminants by reducing the proportion of methane produced by ruminal fermentation and increasing the proportion of proprionic acid in the bovine rumen fluid. Most of the ionophore growth factors are substantially water-insoluble so that a formulation which allows the ionophore to be administered in the drinking water of poultry, cattle, or pigs would be highly beneficial. The water-insoluble nature of these ionophore growth factors makes it difficult to encourage the animals to obtain sufficient concentrations of the growth factors by current administration methods, e.g., as a coating on foodstuffs. Even if a sufficient level is attained, this will vary widely on a day-to-day basis depending on the amount of fluid consumed by the animal. However, most animals drink substantially constant amounts of liquid so that an aqueous-based formulation which can be incorporated into the drinking water would give a more constant level of the ionophore. The constant level is of some importance when the macrolides are used as a growth enhancer, e.g., in the treatment of animal growth but is of paramount importance when the antibiotic properties, e.g., anti-swine dysentery activity, are most significant.
Current ionophore growth factor compositions are primarily coating on feed, e.g., cereal products, as well as other grains and grasses. In addition, slow release, intra-ruminal pellets or boluses have been tried to give proper dosages to animals. For example, see U.S. Pat. No. 4,279,894, issued July 21, 1981. However, although the use of liquid carriers have been contemplated, the water-insoluble properties of most ionophore growth factors have made this an unrealistic possibility. Therefore, a carrier of the ionophore growth factor is necessary for aqueous formulations.
Lipid vesicles have not been considered particularly good carriers for water-insoluble materials such as ionophore growth factors because their cost has been too high for use in animal feed and the instability of the lipid vesicles when carrying large quantities of lipophilic material. Lipid vesicles are substantially spherical structures made of materials having a high lipid content, e.g., surfactants or phospholipids. The lipids of these spherical vesicles are organized in the form of lipid bilayers. The lipid bilayers encapsulate an aqueous volume which is either interspersed between multiple onion-like shells of lipid bilayers (forming multilamellar lipid vesicles or "MLV") or the aqueous volume is contained within an amorphous central cavity. The most commonly known lipid vesicles having an amorphous central cavity filled with aqueous medium are the unilamellar lipid vesicles. Large unilamellar vesicles ("LUV") generally have a diameter greater than about 1.mu. while small unilamellar lipid vesicles ("SUV") generally have a diameter of less than 0.2.mu.. There are a variety of uses for lipid vesicles including the use as adjuvants or as carriers for a wide variety of materials.
Although substantially all the investigation of lipid vesicles in recent years has centered on multilamellar and the two types of unilamellar lipid vesicles, a fourth type of lipid vesicle, the paucilamellar lipid vesicle ("PLV"), exists. This lipid vesicle has barely been studied heretofore and has only been manufactured previously with phospholipids. PLV's consist of about 2 to 10 peripheral bilayers surrounding a large, unstructured central cavity. In all the previously described PLV's, this central cavity was filled with an aqueous solution. See Callo and McGrath, Cryobiology 1985, 22(3), pp. 251-267.
Each type of lipid vesicle appears to have certain uses for which it is best adapted. For example, MLV's have a higher lipid content than any of the other lipid vesicles so to the extent that a lipid vesicle can carry a lipophilic material in the bilayers without degradation, MLV's have been deemed more advantageous then LUV's or SUV's for carrying lipophilic materials. In contrast, the amount of water encapsulated in the aqueous shells between the lipid bilayers of the MLV's is much smaller than the water which can be encapsulated in the central cavity of LUV's, so LUV's have been considered advantageous in transport of aqueous material. However, LUV's, because of their single lipid bilayer structure, are not as physically durable as MLV's and are more subject to enzymatic degradation. SUV's have neither the lipid or aqueous volumes of the MLV's or LUV's but because of their small size have easiest access to cells in tissues.
PLV's, which can be considered a sub-class of the MLV's, are a hybrid having features of both MLV's and LUV's. PLV's appear to have advantages as transport vehicles for many uses as compared with the other types of lipid vesicles. In particular, because of the large unstructured central cavity, PLV's are easily adaptable for transport of large quantities of aqueous-based materials. Also as illustrated in previously cited U.S. patent application Ser. No. 157,571 now U.S. Pat. No. 4,911,928, the aqueous cavity of the PLV's can be filled wholly or in part with an apolar oil or wax and then can be used as a vehicle for the transport or storage of hydrophobic materials. The amount of hydrophobic material which can be transported by the PLV's with an apolar core is much greater than can be transported by MLV's. The multiple lipid bilayers of the PLV's provides PLV's with additional capacity to transport lipophilic material in their bilayers as well as with additional physical strength and resistance to degradation as compared with the single lipid bilayer of the LUV's.
All of the early lipid vesicle or liposome studies used phospholipids as the lipid source for the bilayers. The reason for this choice was that phospholipids are the principal structural components of natural membranes. However, there are many problems using phospholipids as artificial membranes. First, isolated phospholipids are subject to degradation by a large variety of enzymes. Second, the most easily available phospholipids are those from natural sources, e.g., egg yolk lecithin, which contain polyunsaturated acyl chains that are subject to autocatalyzed peroxidation. When peroxidation occurs, the lipid structure breaks down, causing premature release of encapsulated materials and the formation of toxic peroxidation byproducts. This problem can be avoided by hydrogenation but hydrogenation is an expensive process, thereby raising the cost of the starting materials. Cost is a third problem associated with the use of phospholipids on a large scale. A kilogram of egg yolk lecithin pure enough for pharmacological liposome production presently costs in excess of $1,000. This is much to high a cost for a starting material for most applications. Even less highly purified phospholipids are too expensive for most animal uses.
Recently, there has been some indication, particularly from L'Oreal and Micro Vesicular Systems, Inc., that commercially available surfactants might be used to form the lipid bilayer in liposome-like multilamellar lipid vesicles. Both surfactants and phospholipids are amphiphiles, having at least one lipophilic acyl or alkyl group attached to a hydrophilic head group. The head groups are attached to one or more lipophilic chains by ester or ether linkages. Commercially available surfactants include the Brij family of polyoxyethylene acyl ethers, the SPAN sorbitan alkyl esters, and the TWEEN polyoxyethylene sorbitan fatty acid esters, all available from ICI Americas, Inc. of Wilmington, Del.
The methods and materials disclosed herein for producing the paucilamellar lipid vesicles all yield vesicles with a high aqueous or oil volume. Electron micrographs confirm that the paucilamellar lipid vesicles are distinct from the LUV's and the classic MLV's.
Accordingly, an object of the invention is to provide an aqueous-based formulation of an ionophore growth factor.
Another object of the invention is to provide a formulation having factor encapsulated within a nonphospholipid vesicle.
A further object of the invention is to provide a method of preparing an aqueous-based formulation of a substantially water-insoluble ionophore growth factor which exhibit both growth promoting and antibiotic action.
A still further object of the invention is to provide a method of treatment of animals to enhance growth and provide antibiotic action.
These and other objects and features of the invention will be apparent from the following description.